The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference in their entirety for all that they disclose, and for convenience are referenced in the following text by reference number and are listed by reference number in the appended bibliography.
Recombinant DNA manipulation and genetic engineering can improve crop performance by increasing yield, biomass, and/or tolerance to abiotic or biotic stress. To maximize crop performance the foreign gene needs to be expressed, and the corresponding gene product (RNA or peptide) needs to accumulate, at a specific place and time. Promoters and terminators control developmental, tissue, and temporal gene expression, as well as RNA processing and stability. Together the promoter and terminator can be used to enhance the agronomic, nutritional or pharmaceutical value of a plant or crop.
Promoters
The promoter is usually located upstream (5′) of the initiation or start codon (ATG) of the gene (FIG. 1). The in silico identification of putative promoters can be conducted by the recognition of regulatory motifs, such as TATA boxes, TC-motifs, cis-regulatory elements, or core promoter structures (1-4). In addition, putative promoters can also be identified by their physical location to a gene. Genes can be identified by searching genome databases or by scanning databases (5, 6).
In general, the regulatory sequences for an RNA polymerase II-dependent promoter reside in the region approximately 2900 to 35 basepairs (bp) up-stream of the initiation or start codon (ATG) of the gene. For example, the full-length promoter for ACC oxidase from peach is 2919 bp (7), the full-length promoter for cytokinin oxidase from orchid is 2189 bp (8), the full-length promoter for glucuronosyltransferase from cotton is 1647 bp (9), full-length promoter for glutathione peroxidase1 from Citrus sinensis is 1600 bp (10), and the full-length promoter for the nodule-enhanced PEP carboxylase from alfalfa is 1277 bp (11). The convention for specifying the position of the nucleotides in a promoter is to identify the number of by prior to the “A” in the start codon. The nucleotide preceding the start codon is designated as −1. The functional regions of a plant promoter are typically between −1700 to −1 bp. However, functional plant promoters can be located between −200 to −1 bp, −500 to −1 bp, −1000 to −1 bp, −1500 to −1 bp, and −2000 to −1 bp.
As mentioned above, the promoter contains the 5′ untranslated region (5′UTR) of the transcribed RNA. The 5′UTR may play an important role in the expression of the transcript (12-16) by controlling transcription (17) and RNA stability (18). The 5′UTR (could also control the efficiency of translation (19, 20).
There are many types of promoters. Constitutive promoters provide continuous expression and can vary in strength of expression from weak to strong. They can be used for applications that include, but are not limited to, testing the effects of a gene construct with a selectable marker such as antibiotic, herbicide or chemical resistance or for expression of an antisense construct, RNAi, or a foreign gene. Non-constitutive promoters do not continuously produce transcript or RNA. Non-constitutive promoters may induce or increase transcription of genes in response to a signal, such as an environmental cue or other stress signal including biotic and/or abiotic stresses. Non-constitutive promoters include developmentally preferred promoters, tissue-preferred promoters, tissue-specific promoters, cell-type-specific promoters, and inducible promoters. Developmentally preferred promoters limit expression to specific developmental stages. Tissue-specific or tissue-preferred promoters limit expression to specific cells or tissues that include, but is not limited to, fiber-specific, green tissue-specific, root-specific, stem-specific, flower-specific, vascular-specific, xylem-specific or phloem-specific promoters. Inducible promoters initiate expression in response to stimuli, including, but not limited to, mechanical manipulation, heat, cold, salt, flooding, drought, salt, anoxia, pathogens, such as bacteria, fungi, and viruses, and nutritional deprivation.
Terminators
Terminators are typically located downstream (3′) of the gene, after the stop codon (TGA, TAG or TAA). Terminators play an important role in the processing and stability of RNA as well as in translation. Most, but not all terminators, contain a polyadenylation sequence or cleavage site. Examples of specific polyadenylation sequences are AAUAAA or AAUAAU. These sequences are known as the near upstream elements (NUEs) (21). NUEs usually reside approximately 30 bp away from a GU-rich region (22-24), known as far upstream elements (FUEs). The FUEs enhance processing at the polyadenylation sequence or cleavage site, which is usually a CA or UA in a U-rich region (25). Within the terminator, elements exist that increase the stability of the transcribed RNA (26-28) and may also control gene expression (29, 30).
Monocot and Dicot Regulatory Sequences
Some promoters work in both dicotyledonous (dicot) and monocotyledonous (moncot) plants (31), and others, such as chimeric promoters, have been engineered to work in both classes of flowering plants (32). The typical case is that dicotyledonous regulatory sequences control expression in dicots, and monocotyledonous regulatory sequences control expression in monocots. This is particularly true for promoters.
Thus, there is a need in the art to identify and use new regulatory sequences for utilization in plants. The development of plant regulatory sequences for genetic engineering is important to meet the increased demand for the production of food, fiber, feed, biofuel and bio-based materials and nutraceuticals.